A major obstacle to reversing hair loss disorders and creating fully functional skin replacements to treat skin loss injuries is the lack of an effective means to orchestrate the biological activities among distinct cell populations in the skin. The hair follicle is a self-renewing mini-organ that depends on interactions between epithelial keratinocytes and fibroblasts in their immediate dermal microenvironment for successful development, homeostasis, and repair. It has been demonstrated that dermal papilla cells (DPCs) of growing follicles are the commanding force to regulate the activities of follicular keratinocytes and drive the hair cycle. However, the DP niche is heterogeneous and exhibits hair cycle-associated plasticity. Whether there is an inductive DP subpopulation that programs specific signals to stimulate DP inductivity and guide keratinocyte behaviors to regenerate hair follicles remain poorly defined. The lack of such knowledge represents an important knowledge gap because current clinical approaches to the treatment of hair growth disorders and hair follicle reconstitution in tissue-engineered skin equivalents remain highly inefficient. The long-term goal is to develop improved therapeutic approaches for skin and hair follicle regeneration by elucidating the roles of specialized mesenchymal populations in the hair follicle. The overall objective of the proposed work is to systematically characterize the specific hair-inducing DP niche population during early anagen that guides the EMI activities and hair growth, and assess their ability to promote hair follicle formation from human keratinocytes in engineered skin substitute (ESS). The central hypothesis is that the DP niche contains a dynamic mix of active (inductive) and quiescent (responsive) DPCs, and CD133-positive (CD133+) DPCs function as an inductive center by coordinately generating mesenchymal-mesenchymal and mesenchymal-epithelial signals. This hypothesis is supported by our published data and reports from other groups demonstrating CD133+ DPCs is a unique subpopulation in the DP and possess the strong capability to increase hair inductivity. In the first aim, we will determine the characteristics of CD133+ DPCs in hair follicle neogenesis. We will systematically investigate their specific functions in clonal growth and hair inductivity using in vitro 3D culture and in vivo skin reconstitution assay. In the second aim, we will evaluate the trichogenic ability of CD133+ DPCs in ESS to stimulate the induction of hair follicle growth from human keratinocytes and human DPCs. This work will provide ample information to produce a new and in-depth understanding of the DP niche in hair follicle regenerative growth and clinical relevance of CD133+ DPCs in skin tissue engineering. The findings are expected to close a major gap in hair follicle biology by elucidating the initial hair-inducing signals from the dermal niche to promote hair follicle regeneration. These outcomes are significant because they may disclose the unknown biological basis of the skin microenvironment, and have an important positive impact on human health by opening the possibilities of developing effective strategies for patients with life-threatening skin wounds or disfiguring hair loss.